Autophagy orchestrates adaptive responses to targeted therapy in endometrial cancer

Abstract

Targeted therapies in endometrial cancer (EC) using kinase inhibitors rarely result in complete tumor remission and are frequently challenged by the appearance of refractory cell clones, eventually resulting in disease relapse. Dissecting adaptive mechanisms is of vital importance to circumvent clinical drug resistance and improve the efficacy of targeted agents in EC. Sorafenib is an FDA-approved multitarget tyrosine and serine/threonine kinase inhibitor currently used to treat hepatocellular carcinoma, advanced renal carcinoma and radioactive iodine-resistant thyroid carcinoma. Unfortunately, sorafenib showed very modest effects in a multi-institutional phase II trial in advanced uterine carcinoma patients. Here, by leveraging RNA-sequencing data from the Cancer Cell Line Encyclopedia and cell survival studies from compound-based high-throughput screenings we have identified the lysosomal pathway as a potential compartment involved in the resistance to sorafenib. By performing additional functional biology studies we have demonstrated that this resistance could be related to macroautophagy/autophagy. Specifically, our results indicate that sorafenib triggers a mechanistic MAPK/JNK-dependent early protective autophagic response in EC cells, providing an adaptive response to therapeutic stress. By generating in vivo subcutaneous EC cell line tumors, lung metastatic assays and primary EC orthoxenografts experiments, we demonstrate that targeting autophagy enhances sorafenib cytotoxicity and suppresses tumor growth and pulmonary metastasis progression. In conclusion, sorafenib induces the activation of a protective autophagic response in EC cells. These results provide insights into the unopposed resistance of advanced EC to sorafenib and highlight a new strategy for therapeutic intervention in recurrent EC.

Keywords: MAPK/JNK; PDX; autophagy; endometrial cancer; kinase; lysosomes; sorafenib; targeted therapy.

Figure 1.

(see previous page) Sorafenib targets endometrial cancer cells with high specificity and induces apoptotic cell death. (A) Analysis of sorafenib effects in a high-throughput screening using kinase inhibitors in a panel of 494 cancer cell lines Sensitivity was calculated as the fraction of viable cells relative to untreated controls following treatment with 2μM sorafenib for 72 h²¹. The sensitivity values of cell lines are plotted as a function of the ranking of the sensitivity (from sensitive to resistant). Uterus cell lines (red circles) are over-represented among sensitive cell lines. (B) Left, ranking and sensitivity of _EC cells to sorafenib among the 494 cancer cell lines. Right, graphical representation of the ranks, according to decreasing order of sensitivity, among the 494 cell lines analyzed. EC cells are represented as red lines in the top half of the ranking. Thick bar represents the median rank. (C) Box plot illustrating sorafenib effects in the uterus (n cell lines = 7) compared with the rest of the tissues (18 other tissues, n cell lines = 487). P-value = 6.96e-7 (FDR<1e-5, t-test). For additional information please see Fig. S2B. Other tissues include bladder, skin, bone, brain, lung, stomach, kidney, thyroid, ovary, pancreas, breast, esophagus, cervix, intestine and liver. Sorafenib effects were assessed in vitro by measuring p-EIF4E levels by western blot in a dose-dependent treatment (D) and a time-course treatment (E) in Ishikawa cells. Results were further validated in 2 independent EC cell lines (HEC-1A and RL95–2) at final concentration (F). Western blot against tubulin was performed to ensure equal protein loading amounts. MTT assays in Ishikawa, HEC-1A and RL95–2 cells (G) and clonogenicity assays in Ishikawa cells (H) were performed to measure sorafenib effects on cell viability. Values are represented as the percentage of viable cells or colonies compared with untreated cells. Sorafenib-induced apoptosis after 24 h of treatment was characterized by measuring DNA fragmentation by flow cytometry (sub G₁ phase) (I), quantifying pyknotic nuclei by Hoechst staining (J) and analysis of ANXA5/Annexin V-positive cells by flow cytometry (K) in Ishikawa cells. (L) Western blot showing activation of inducer CASP9, executioner CASP3 and PARP cleavage after sorafenib 20μM treatment of 24 h in different EC cell lines. All experiments were performed in triplicate. T test statistical significant differences were calculated by comparison to untreated conditions. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 100μM.

Figure 2.

(see previous page) Sorafenib treatment activates an autophagic flux. (A) Pearson's correlation coefficients (Y-axis) between gene expression and sorafenib sensitivity of 20 EC cell lines are plotted as a function of the ranking of the coefficients (X-axis). Each data point represents a gene. Gene set enrichment analysis shows lysosomal genes (red circles) are enriched among those with negative correlation between expression and sorafenib sensitivity. (B) Representative western blot and densitometry quantification from 3 independent experiments showing increased LC3B-II after sorafenib (20μM) treatment of 12 h in Ishikawa, HEC-1A and KLE _EC cells. Western blot against tubulin was performed to ensure equal protein loading amounts. (C) 12-h sorafenib treatment causes an increase in immunofluorescent LC3B-II puncta per cell that is further increased when sorafenib is combined with CQ, reflecting an autophagic response in Ishikawa and HEC-1A _EC cells. Left, representative immunofluorescent images of Ishiwaka cells. Scale bar: 50 µm. Right, quantifications are represented as percentage of total cell population. Statistical values (t-test) compare the number of LC3B-II puncta per cell between conditions. Autophagic flux arrest using 2 different concentrations of CQ (D) and bafilomycin A₁ (E). Ishikawa cells were lysed after 24 h of treatment and levels of SQSTM1 were analyzed by western blot. Western blot against tubulin was performed to ensure equal protein loading amounts. Densitometry quantifications of SQSTM1 from 3 independent experiments are also shown. (F) Autophagic flux analysis. Left, representative immunofluorescent images of Ishiwaka cells transfected with a chimeric mRFP-GFP-LC3B probe showing mRFP, GFP and merged mRFP and GFP (yellow) puncta. Scale bar: 15 µm. Right, quantification of red (mRFP⁺ GFP⁻) and yellow (mRFP⁺ GFP⁺) puncta per cell. (G) Left, schematic illustration of autophagic process with the most relevant autophagic structures. Right, representative transmission electron microscopy (TEM) images showing formation of phagophores (P), autophagosomes (AP) and autolysosomes (AL) after sorafenib (20μM) treatment of 24 h. Also, quantification of increased P, AP and AL. 100 cells in each condition were quantified using this method (n = 3). Asterisks indicate vacuolization and dilated ER cisternae. N, nucleus. (H) Left, representative micrographs of 3D cultures treated with sorafenib showing decreased cytoplasmic content and the presence of autophagic organelles. Ishikawa cells were cultured in matrigel to form 3D organotypic structures. 3D cultures were left untreated or treated with sorafenib (20μM) for 24 h and subsequently processed for TEM analysis. M, mitochondria. Right, 3D cultures were additionally processed for western blot against LC3B-II. LC3B-II densitometry quantification from 3 independent experiments are also shown. (I) Left, TEM representative micrographs illustrating autophagy activation in response to sorafenib in vivo in HEC-1A subcutaneous tumors injected in SCID mice. Images show a drastic depletion of cytoplasmic subcellular compartments and the presence of autophagic structures. L, lipid droplet. RER, rough endoplasmic reticulum. Xenografts were left to grow for 11 d and mice were injected intraperitoneously with sorafenib 15 mg/kg/d. At the end of the treatment, tumors were collected and processed for TEM. Right, tissue immunohistochemistry for LC3B (dilution 1:100) and SQSTM1 (dilution 1:3000). All experiments were performed in triplicate.

Figure 3.

Sorafenib induces autophagy through ER stress and MAPK8/9/10 activation. (A) TEM representative micrographs showing dilated ER cavities in sorafenib-treated Ishikawa cells for 24 h. For additional information please see Fig. S4A. (B) Left, representative images showing increased and discontinuous fluorescence intensity in endoplasmic reticulum upon sorafenib treatment. Untreated and sorafenib-treated Ishikawa cells were cultured in the presence of ER-Tracker Blue-White DPX and analyzed at 4 h post-treatment. Scale bar: 50 µm. Right, quantification of ER-Tracker Blue-White DPX intensity according to refs. and using ImageJ software. (C) Left, schematic illustration representing 2 characteristic UPR responses, EIF2AK3/PERK-ATF4 and ERN1/IRE1. Right, XBP1 splicing analysis by PCR and enzymatic restriction and DDIT3/CHOP protein levels by western blot with densitometry quantification in Ishikawa cells treated with sorafenib. Splicing of XBP1 mRNA results in the excision of a 26-nucleotide intronic region, causing a frame shift in the coding sequence and the removal of a PstI restriction site. Therefore, PstI can digest unspliced XBP1 mRNA (XBP1u), generating XBP1 u1 and u2 fragments, but not spliced XBP1 mRNA (XBP1s). h represents a hybrid band composed of XBP1u and XBP1s single-stranded DNA produced during PCR. Western blot against tubulin was performed to ensure equal protein loading amounts. (D) Kaplan-Meyer and (E) disease-free survival curves comparing the outcome of EC cases with or without overexpression of MAPK8/JNK1, MAPK9/JNK2 and MAPK10/JNK3. A z-score≥ 3 was used as threshold for increased expression. Data were extracted from TCGA_ucec (RNAseq_V2). (F) Western blot and densitometry quantification (n = 3) showing activation of MAPK8/9/10 and its target JUN after a time course treatment of sorafenib (20 µM) in Ishikawa cells. (G) Activation of MAPK8/9/10 and JUN by western blot in sorafenib-treated primary EC biopsies. CEP-1347 inhibits activation of MAPK8/9/10 and JUN (H) and blocks LC3B-II increase in Ishikawa _EC cells (I). Densitometry quantifications from 3 independent experiments are also shown. (J) Representative western blot and densitometry quantification (n = 3) showing impaired LC3B-II accumulation in mapk8/9^−/− cells after sorafenib treatment. (K) Representative immunofluorescence images and quantification of the number of LC3B-II puncta per cell in wild-type and mapk8/9^−/− MEFs treated with sorafenib or sorafenib combined with CQ. Scale bar: 50 µm. (L) Analysis of SQSTM1 protein levels by western blot in wild type and mapk8/9^−/− MEFs showing that CQ and Mapk8/9-targeted deletion abrogates SQSTM1 proteolysis in response to sorafenib in wild-type and mapk8/9^−/− MEFs. Western blot against tubulin was performed to ensure equal protein loading amounts. SQSTM1 densitometry analysis is also shown. All experiments were performed in triplicate.

Figure 4.

(see previous page) Autophagy inhibition potentiates sorafenib anticancer properties. (A) MTT cell viability assay in sorafenib (SOR)-treated Ishikawa and HEC-1A cells in combination with chloroquine (CQ) for 12 h. Values are represented as the percentage of viable cells compared with untreated cells. (B) Quantification of percentage of pyknotic nuclei by Hoechst staining of Ishikawa and HEC-1A cells treated with sorafenib in combination with CQ for 12 h. (C) Analysis of ANXA5/Annexin V-positive Ishikawa cells by flow cytometry after sorafenib treatment combined with CQ for 12 h. Values are expressed as percentage of cells in relation to total number of cells. (D) Left, western blot and densitometry quantification (n = 3) showing decreased BECN1 expression in BECN1-shRNA infected Ishikawa (IK) cells. Western blot against tubulin was performed to ensure equal protein loading amounts. Ishikawa cells transduced with lentiviral particles encoding shRNA-scrambled or shRNA-BECN1 and selected with puromycin. Right, quantification of apoptotic nuclei after 2 doses of Sorafenib treatment of 24 h in shRNA-scrambled Ishikawa cells and cells with constitutive decreased expression of BECN1. (E) Western blot showing increased activation of inducer CASP9 and executioner CASP3 in shRNA-BECN1 Ishikawa cells after sorafenib (20 µM) treatment of 24 h. (F) HEC-1A subcutaneous tumors. Fourteen d after HEC-1A injection, mice were randomized and treated with vehicle (n = 5), CQ 60 mg/kg (n = 5), sorafenib 15 mg/kg (n = 5) and sorafenib plus CQ (n = 5) and processed at d 29. Top-left, representative image of subcutaneous tumors after treatment. Top-right, subcutaneous tumor growth kinetics. No differences in tumor size were noticed between vehicle and CQ (1.41 ± 0.21cm, 0.90 ± 0.08cm; p = 0.221), when comparing vehicle with sorafenib (1.41 ± 0.21cm, 1.15 ± 0.19cm; p = 0.161) or between CQ and sorafenib (0.90 ± 0.08cm, 1.15 ± 0.19cm; p = 0.447). Notably, combination of sorafenib with CQ (0.25 ± 0.04cm³) led to a marked reduction in tumor volume in all mice (p < 0.001) when compared with either agent alone. Values are represented as tumor volume fold increase in relation to d 4 after injection. Down, tissue immunohistochemistry for LC3B (dilution 1:100) and SQSTM1 (dilution 1:3000). All experiments were performed in triplicate. (G) Inhibition of metastatic EC growth by sorafenib combined with CQ. _EC cells MFE-296 were stably infected with EGFP-luciferase. After selection, 50×10⁴ MFE-296 cells were retro-orbitally injected and 11 d after injection mice were treated with vehicle (n = 5), CQ 60 mg/kg (n = 5), sorafenib 15 mg/kg (n = 5) and sorafenib plus CQ (n = 5) until d 21 (see Materials and Methods for further details). Left, representative bioluminescence imaging comparing progression of metastasis in lungs at 15 and 21 d after injection of MFE-296 cells. Right, quantification of the relative bioluminescence intensity. Values are expressed as fold increase in relative luciferase units (RLU) compared with time 0 (11 d post-injection). (H) Kaplan-Meyer survival curve of mice retro-orbitally injected with MFE-296 cells and treated until d 21. At d 21, treatment was terminated and mice were monitored twice a day. (I) Representative lung morphology analysis by hematoxylin and eosin staining. Samples were harvested at d 21 based on bioluminescent and survival analysis (n = 5/condition). (J) Quantification of metastases diameter.

Figure 5.

Autophagy inhibition potentiates sorafenib cytotoxicity in orthotopic patient-derived xenotransplants. (A) Schematic representation of patient-derived orthoxenotransplant implants procedure and treatment. EEC tumors were surgically removed and small pieces were implanted in recipient female mice. Once engrafted, tumors were propagated to a cohort of 20–45 mice, randomized and treated accordingly. Response of engrafted grade-I END72X (B), grade-II END82X (C) and grade-III END90X (D) tumors after control, sorafenib (30 mg/kg), CQ (60 mg/kg) and sorafenib plus CQ treatments. Animals were treated through 21 d (see Materials and Methods). Graphs illustrate responses of 3 endometrial orthoxenografts of different histological degree after 21 d of treatment. Representative images of endometrial-engrafted tumors are also shown.